Applications of transgenic and knockout mice in alcohol research.

Multiple genetic and environmental factors contribute to the development of alcoholism. Researchers attempting to elucidate the roles of specific genes in alcoholism risk have benefited from advances in genetic engineering. Two important tools used by researchers include transgenic mice, in which a foreign gene is integrated into an animal's genetic material, and knockout/knock-in mice, in which targeted genes either are rendered nonfunctional or are altered. Both of these animal models are currently used in alcohol research to determine how genes may influence the development of alcoholism in humans.

A lcoholism is a complex disord e r that encompasses seve r a l p h y s iological and behavioral characteristics (also re f e r re d to as phenotypes), including atypical responses to alcohol in initial sensitivity, tolerance, consumpt i o n , and withdrawal as well as vulnerability to the rew a rding effects of alcohol. Human and animal studies have both s h own that these aspects of alcoholism a re often mediated by chemical pathways in the brain, known as neurotransmitter systems (Diamond and Go rdon 1997;Harris 1999). A major goal for neuroscientists has been to identify genes and proteins in the brain that influence the expression of alcoholism. In c reased knowledge of chemicals in the brain and receptor pro t e i n s (i.e., protein molecules that re c o g n i ze and bind neurotransmitters) has provided scientists with a priori reasons for studying specific genes and pro t e i n s , which are sometimes re f e r red to as cand i d a t e genes and proteins. For example, alcohol affects nearly all brain activity, including enhancement of inhibition, mediated by the gamma-aminobutyric acid (GABA) neurotransmitter system; a c t i vation of rew a rd pathways that are dopaminergic (DA) and sero t o n e r g i c ( 5 -H T); and effects on enzyme proteins that are located inside nerve cells (i.e., neurons) (Di a m o n d and Go rd o n 1997; Grobin et al. 1998). There f o re , these systems (as well as several others, including nonneuronal genes) have p roduced many candidate genes to i n vestigate for their roles in the deve lopment of alcoholism.
One approach that is part i c u l a r l y e f f e c t i ve in the hunt for candidate genes is the re verse genetics appro a c h , in which a gene of interest is altered to change its expression, or function, in t h e e n t i re animal. This approach invo l ve s generating transgenic mice, in which a f o reign gene is integrated into an anim a l's genetic material, as well as knockout or knock-in mice, in which candi-date genes are either inactivated or altere d , resulting in a lack of or change in protein expression. Although mouse models may seem poor re s e a rch tools for studying the genetics of human neuro p h y s i o l o g y and behavior, substantial genetic similarities exist betwe e n mice and humans, and known correspondence exists b e t ween mouse c h romosomal re g i o n s and human chromosomes (Si l ver 1995).
This article describes the generation of transgenic mice and knockout/ knock-in mice and recent examples of h ow these techniques have been applied in alcohol re s e a rc h .

Transgenics
In the context of mouse models, the term transgenic refers to the intro d u ction of a fore i g n 1 gene (known as a transgene) into the genetic material of a mouse in both the re p ro d u c t i ve (i.e., germ) cells and the nonre p ro d u c t i ve (i.e., somatic) cells. This process leads to the expression and propagation of t h e gene across future generations. Of t e n t h e purpose of this technique is to create mice that express more than normal amounts of the gene product (i.e., protein). In some applications, howe ve r, the scientific goal is to introduce a differe n t form of the gene in question. This technique allows re s e a rc h e r s to evaluate the role of specific genes in the deve l o pment of disease. For example, a line of transgenic mice generated to study alcoholism would carry a gene that is know n or suspected to have a role in some aspect of the disease. Re s e a rchers can then study the animals' behaviors to e valuate the role of the gene in alcoholism.

Creating Transgenic Mice
Basic re q u i rements for creating a transgenic mouse include (1) identifying and isolating the candidate gene of intere s t f rom its original organism (i.e., from the DNA of the organism's cells) and (2) selecting a suitable promoter that is placed adjacent to the transgene. Pro m o t e r s a re stretches of DNA associated with a specific gene that guide the expre s s i o n of the gene to specific areas in the brain and turn the expression of the gene "o n" either before birth (i.e., pre n a t a l l y ) or after birth (i.e., postnatally). The choice of promoter by the scientist depends on its location in the brain and the time in which (i.e., prenatal or postnatal) the transgene must be e x p ressed. For example, the pro m o t e r for the α-calmodulin kinase II (αC a m K I I ) enzyme gene directs postnatal expre ssion to the forebrain. There f o re, any gene associated with this promoter would be found in the forebrain after birt h .
Once the transgene construct (i.e., the promoter and DNA) is pro d u c e d , many copies are introduced into fert i li zed mouse eggs (i.e., embryos) at the single-cell stage (see figure 1). To cre a t e mouse embryos, female mice are hormonally induced to hyperovulate and then mated to males for fert i l i z a t i o n . The fert i l i zed eggs are harvested fro m the female and injected with the transgene. The injection takes place early after f e rtilization, when the embryo contains two sets of DNA, one from each pare n t . Each set of DNA exists in a separate s t ru c t u re, called a pronucleus; there f o re , both male and female pronuclei exist. Copies of the promoter and foreign gene c o n s t ruct are microinjected directly into the male pronucleus with a fine glass needle. The re s e a rcher injects the transgene before the first cell division to ensure that the DNA will develop in all cells of the adult animal. The pronuclei then fuse, and the cell begins to divide normally.
Ap p roximately 50 to 90 percent of the eggs surv i ve the p ro c e d u re and are implanted into the oviducts of foster m o t h e r s , w h e re the embryos develop to term. Mice that carry the transgene, identified using DNA analysis techniques, are called progenitor or founder mice. These mice are bred with n o ntransgenic mice, and the offspring are tested for the presence of the transgene to confirm that the transgene has integrated into the germ cells. If the integration has occurred, subsequent b re e d i n g continues between mice carrying the transgene, re f e r red to as the F 1 g e n e r ation, producing lines of transgenic mice ( F 2 generation). For more information on creating transgenic mice, see Camper 1987 and Picciotto and Wickman 1998.
Although re s e a rchers can contro l the expression of the transgene with the choice of pro m o t e r, one limitation of the transgenic technique is its inability to target the integration of the transgene to its natural location on the chro m osome. The site of integration is unique for each microinjection, and the transgene can be randomly inserted anywhere on any chromosome. This outcome can result in the disruption of a sequence The source of this gene could be mouse or another mammalian species, including human.

Figure 1
General procedure for the generation of transgenic mice.
Foreign gene is injected into the cellular structure containing the genetic material from the father.
Embryo is transferred into a foster mother for embryonic development.
Mouse pups (i.e., F 1 animals) carrying and expressing the f o r e i g n gene are identified. Each offspring (i.e., F 2 animal) carries the foreign gene in all of its cells.
of one of the host animal's own genes (i.e., known as insertional mutagenesis), p roducing changes in behavior that could mistakenly be attributed to the transgene itself. Also, the number of integrated copies of the transgene cannot be c o n t rolled, and having more copies of a gene does not necessarily i n d i c a t e i n c reased ove re x p ression of the gene.
To control for this, the existence of m o re than one founder and consequently more than one line of mice for each transgene is desirable. The site of integration and level of expression will differ in each founder, and transgenic mice that descend from the same founder will share the same chro m o s omal integration site. If each transgenic line displays the same changes in behavi o r, it is more likely that it is attributable to the transgene.

Using Transgenic Mice in Alcohol Research
Transgenic mice have traditionally been used to study deve lopmental pro c e s s e s and as models of human diseases. Although transgenic lines have been generated for s e veral genes, their use has been somewhat limited in alcohol re s e a rch. Howe ve r, this application remains useful for identifying candidate genes that underlie specific aspects of alcoholism (We h n e r and Bowers 1995). For example, animal studies have shown that the sero t o n e rgic (5-HT) neurotransmitter system is i n vo l ved in alcohol consumption and other alcohol-related behaviors (Li and Mc Bride 1995). There f o re, Engel and colleagues (1998) created transgenic mice ove re x p ressing the 5-HT 3 re c e p t o r p rotein to investigate its role in alcohol and other drug abuse. The re s e a rc h e r s used the αCaMKII promoter to dire c t e x p ression of the 5-HT 3 transgene to the forebrain. Ex p ression of the 5-HT 3 receptor was greatly increased in the transgenic mice; receptor binding of a 5-HT 3 agonist (i.e., a chemical that mimics serotonin at the 5-HT 3 s u btype) was increased nearly a hundre dfold in cortical regions of the brain in one of four transgenic lines tested. The remaining lines exhibited lower leve l s of receptor expre s s i o n .
The transgenic mice we re tested for alcohol drinking (Engel et al. 1998) and initial sensitivity to alcohol (En g e l and Allan 1999). Results indicated that ove re x p ression of 5-HT 3 re c e p t o r s d e c reased alcohol consumption by 46 p e rcent. In support of the role of 5-HT 3 e x p ression in alcohol consumption, Engel and colleagues (1998) re p o rt e d that the level of alcohol consumption in the four transgenic lines was re l a t e d to their levels of receptor ove re x p re ssion (i.e., the greater the level of ove re x p ression, the greater the reduction in consumption). In contrast, ove re x p re ssion of 5-HT 3 receptors increased sensitivity to the activating effects of low doses of alcohol. When some mice are a d m i n i s t e red low doses of alcohol, their locomotor activity increases. This phenomenon is re f e r red to as alcohol-induced hyperlocomotion and is a measure used in mice to test for initial sensitivity to alcohol. The authors have suggested that 5-HT 3 receptors play a role in i n c reased initial sensitivity that may be related to decreased alcohol consumption. The GABA neurotransmitter system p rovides another a venue for using transg e n i c s in alcohol re s e a rch. The GABA A receptor family consists of at least 16 diff e rent protein molecules (i.e., subunits) assembled 5 at a time, creating a re c e ptor complex that surrounds a channel. When GABA or GABA-like compounds bind to the receptor and activate it, this channel temporarily opens and allows the passage of negative l y charged molecules (i.e., chloride ions) to pass from the c e l l's exterior to its interior. This ion flow d e c reases the cell's exc i t a b i li t y, which re s u l t s in inhibition. The GABA system is the p r i m a ry modulator of inhibition in the brain (Ba r n a rd et al. 1998). Alcohol enhances the inhibitory effects of GABA at the GABA A receptor; this interaction of alcohol and the receptor appears to cause alcohol's intoxicating and sedating effects.
Nu m e rous studies have established that acute and chronic behavioral effects of alcohol are differentially mediated t h ro u g h G A B Aergic receptor subunits (for a re v i ew, see Grobin et al. 1998). The γ2 subunit, which exists in both a long (γ2L) and short (γ2S) version, has been studied for its role in GABAe r g i c sensitivity to alcohol. Although initial in vitro (i.e., in a test tube) studies inve st i g a t i n g the role of the γ2L subunit in alcohol potentiation of GABAe r g i c function s h owed that this subunit was n e c e s s a ry for alcohol sensitivity; l a t e r studies have not found an absolute γ2 L re q u i rement (Grobin et al. 1998;Sa p p and Yeh 1998). The inconsistencies in these results may be attributable to diff e rences in the in vitro pre p a r a t i o n s used by the inve s t i g a t o r s .
To further investigate the function of the γ2 subunit in a whole animal model, Wick and colleagues (2000) c reated transgenic mouse lines ove re xp ressing the γ2L and γ2S genes. Tw o γ2L and one γ2S lines of transgenic mice we re tested for responses to alcohol, including sedation, ataxia (i.e., loss of coordination), withdrawal seizure s , and acute functional tolerance (AFT) (i.e., a measure of tolerance to alcohol that occurs within one testing session as opposed to tolerance deve l o p m e n t t h a t occurs over several days of alcohol tre a tment and testing).
None of the transgenic lines of mice d i s p l a yed altered responses to alcohol, with the exception of AFT, in which tolerance was decreased in both γ2 L and γ2S transgenic lines compared with nontransgenic mice. The lack of specificity of the γ2L transgene and lack of effects on the other measures may have been caused by insufficient ove re x p re s s i o n of either transgene. Howe ve r, the leve l s of expression we re sufficient to re s c u e the lethal phenotype of gene-targeted mutant mice lacking the entire γ2 gene In other words, mice lacking the entire γ2 gene but ove re x p ressing the γ2 L transgene surv i ved (Bauer et al. 2000).
The 5-HT 3 and γ2L and γ2S transgenic lines are two examples of the use of this technology in alcohol re s e a rc h with a focus on initial sensitivity, tolerance, or consumption. Other transgenic mouse lines have been used to study dive r s e a l c o h o l -related phenotypes, such as alcohol's effects on aggre ss i o n (i.e., transforming growth factor α transgenics [TG Fα]); alcohol as a cofactor in HIV disease (i.e., transactiva t o r p ro t e i n ove re x p ression [Tat]); alcohol's effects on alcohol card i o m yopathy (i.e., alcohol dehyd rogenase transgenics [ADH]); and alcohol-induced neurot oxicity in neonatal cerebellum (i.e., cell re p ressor gene transgenics [bcl-2]) (see table for re f e re n c e s ) .

Gene-Targeting Techniques in Knockout and Knock-in Mice
Knockout and knock-in mice are created by gene-targeting techniques that p roduce animals in which a specific gene has been deleted (i.e., "knocked o u t") or mutated (i.e., "knocked i n" ) . Id e a l l y, by inference, any differences in phenotype observe d in knockout and knock-in mice can be due to the nonfunctional or altered gene. Howe ve r, adaptations during deve l o p m e n t attributable to the mutation and the e x p ression of the background genotype may also produce changes in behavior not directly caused by the missing or mutated gene.
In some instances, when a gene vital to embryonic development is deleted or mutated, knockout or knock-in mice lacking the vital gene cannot deve l o p b e yond a certain stage, demonstrating the gene's re q u i rement for deve l o p m e ntal p rocesses. In this case, the techniques of transgenics and gene-t a r g e t e d mutagenesis can be combined to provide added experimental proof that a deleted or mutated candidate gene is actually the gene responsible for the mutated phenotype. This is accomplished by inserting the wild-type gene (i.e., the normal form of the gene) as a transgene into the host genome of the mutant mouse, as in the γ2 knockout mice previously described. If the re s u l ting phenotype is equivalent to the wild-type phenotype, the mutation is c o n s i d e red "rescued," and pre s u m a b l y the candidate gene is invo l ved in the n e u rochemical pathway of intere s t .

Creating Knockout and Knock-in Mice
To create either knockout or knock-in mice, re s e a rchers use targeted mutagenesis, the site-specific (vs. random) integration of a mutated gene using the c e l l's natural homologous re c o m b i n a t i o n 2 mechanism that occurs during DNA re p l i c a t i o n . In other words, unlike the transgenic techniques, the mutated gene is inserted into its normal location on the chromosome (i.e., the gene is "t a r g e t e d"). This eliminates the pro blems associated with insertional mutagenesis seen in some transgenic lines. Gene-targeting traditionally generates knockout mice in which a candidate gene is re n d e red nonfunctional. Re c e n t l y this technique has been used to cre a t e knock-in mice, in which a mutation is i n t roduced into a candidate gene and the function of the gene is changed but not eliminated.
Re q u i rements of this method include the following: (1) the identification and isolation of the candidate gene fro m mouse DNA and (2) cultured, mouse e m b ryonic stem (ES) cells. ES cells retain the ability to differentiate into all tissues of a developing mouse. Thro u g h genetic engineering techniques, the isolated gene is mutated either to make it nonfunctional or to change its function. The gene is then introduced into the ES cell (see figure 2, p. 182), rather than into an embryo, as in the cre a t i o n of transgenic mice. Once in the ES cell, the mutated gene changes places with the cell's normal (wild-type) gene thro u g h homologous re c o m b i n a t i o n (for a re v i ew, see Capecchi 1994; Picciotto and Wickman 1998). When this occurs, the cell's wild-type gene is disru p t e d and becomes nonfunctional (or altere d if the knock-in strategy is used). ES cells expressing the mutated gene are identified by growing the cells in a petri dish in a specific medium in which only modified cells can surv i ve. Po s i t i ve ES cells are microinjected into 3.5 dayold blastocysts (i.e., fert i l i zed embryo s consisting of 8 to 16 cells). Re s e a rc h e r s implant the blastocysts into foster mothers, where they develop to term. Because the ES cells are introduced at later stages of embryonic cell division, the resulting mouse will be chimeric for the mutated gene (i.e., the mouse will carry the mutated gene in some, but not all, cells.) Fu rther breeding and DNA analysis are re q u i red to identify founder mice, which carry the gene in their germ cells. These mice are used to generate lines of knockout mice. This method does not re q u i re multiple lines of mice, because the mutation does not i n s e rt randomly and only one copy of the mutated gene will have integrated.
Caution is necessary when interpre t i n g results from gene-targeting experiments because of two potentially confounding factors. First, other genes may compensate in response to the disrupted gene, which is nonfunctional thro u g h o u t p renatal and postnatal deve l o p m e n t . Howe ve r, recent advances in gene-targeting techniques that allow re s e a rc h e r s to c o nt rol w h e re and when the mutated gene i s n e u rologically expre s s e d may ove rc o m e this limitation. For example, the deletion of a gene could be programmed to occur in the adult mouse after deve l o pment is complete, there by e l i m i n a t i n g p roblems caused by deve l o p m e n t a l c o mpensation. For more information about these techniques, see Sauer 1998.
The second factor is the mouse's b a c k g round genotype. Because many genes regulate complex behaviors and d rug responses (Ba n b u ry Confere n c e 1997), the expression of any one gene is influenced by the presence of the other genes in an organism's genotype.
T h e re f o re, when a gene is knocked out in a particular inbred strain of mouse (i.e., populations of mice that are genetically identical), the background genotype is not necessarily silent and may mask d i f f e rences in behavior because of the deleted gene. One way to control for this complication is to breed the deleted or mutated gene onto several inbre d strain genetic backgrounds to assess the m o re complicated gene-gene interactions.
In addition, re s e a rchers must consider the possible confounding effects of the genetic background of the original ES cell used for homologous recombination. Some genetic material f rom the ES cell will be closely linked to the region of the mutated version of the gene. During recombination, when the altered gene replaces the wild-type gene, the ES cell's genetic material will be carried with the mutated gene because of its close linkage. This association of ES cell DNA and the targeted gene will not be disrupted, even after seve r a l generations of breeding. If a change in behavior is observed in a gene-targeted mouse, the ES cell's DNA, and not the a l t e red gene, could be the cause. Theref o re, when interpreting behavioral data, re s e a rchers should consider the phenotype of the inbred strain that is the sourc e of the ES cell.

Using Knockout and Knock-in Mice in Alcohol Research
Genes that encode receptor proteins are f requently selected as candidate genes in the gene-targeting approach. Se ve r a l re l e vant receptor subunit knockout mouse lines have been used in alcohol re s e a rch to evaluate contributions of a specific receptor subunit in alcohol's behavioral actions, such as sedation, initial sensitivity, ataxia, withdrawal, tolerance, or consumption. For example, four lines of mice lacking the GABA A receptor subunits α6, β3, γ2L, and δ h a ve been specifically created to test for responses to alcohol and anesthetics ( B owers et al. 2000b;Mihalek et al 1999;Homanics et al. 1997Homanics et al. , 1998Homanics et al. , 1999Quinlan et al. 1998). Su r p r i s i n g l y, mutant mice lacking α6, β3, and γ2 L subunits failed to show different re s p o n s e s to alcohol compared with control mice, as measured by alcohol-induced seda-tion, tolerance, or withdrawal re s p o n s e s (i.e., α6 and γ2L) (Homanics et al. 1997(Homanics et al. , 1998(Homanics et al. , 1999. In addition, alcohol enhancement of GABA A receptor inhibition, alcohol-induced reduction in a n x i e t y, and alcohol-induced hyperlocomotion we re not found to be diff e rent in γ2L mutant mice compare d with control mice (Homanics et al. 1999). Although β3 mutant mice differe d in their response to general anesthetics c o mp a red with nonmutant mice, they did not differ from the contro l mice in alcohol-induced sedation (Quinlan et al. 1998). Explanations for these unexpected results may invo l ve some of the confounding factors previously discussed. For example, the lack of effects in the α6 knockout mice may be caused by i n t e rf e re n c e f rom the mice' s genetic b a c k g round or from ove rc o m p e n s a t i o n by other GABA A subunits. The authors also suggest that the γ2S subunit may substitute for the missing γ2L s u b u n i t , masking a potential role of γ2L in alcohol sensitivity. The amount of alcohol consumed in a free-choice drinking paradigm is one m e a s u re of the re i n f o rcing effects of alcohol. Alcohol consumption was not evaluated in the GABA A s u b u n i t knockout mice previously described; h owe ve r, the δ knockout mice we re tested for alcohol drinking. When δ mutant and wild-type mice we re offere d a choice between water and alcohol solutions ranging from 3 to 11 perc e n t , mice lacking the δ subunit drank significantly less alcohol (Bowers et al. 2 0 0 0b), suggesting that the δ subunit is one factor invo l ved in alcohol pre f e rence. The results of drinking studies using several other receptor knockout mice, some of which are described later in this article, indicate that this behavior is multigenic.
Both the dopaminergic and sero t o nergic neurotransmitter systems have been implicated in alcohol-induced hyperlocomotion and the re i n f o rc i n g effects of alcohol. Receptor proteins for each of these transmitters are derive d f rom families of genes, several of which h a ve been selected as gene-targeting candidates. The D 2 dopamine re c e p t o r knockout mice we re created based on human studies that implicated va r i a n t s of the D 2 receptor in alcoholism, although this association has not been found in eve ry study (Goldman 1995). When D 2 mutant and wild-type cont rol mice we re offered a choice of  et al. 1998). A similar study of D 1 receptor mutants also re p o rted a decre a s e in consumption that may have been associated with higher levels of dopamine in some brain regions (El -Ghundi et al. 1 9 9 8 ) .
De c reased initial sensitivity has been associated with alcoholism in human populations (Schuckit 1988); there f o re , this phenotype is frequently tested in mice using alcohol-induced increases in locomotor activity as the measure of s e n s i t i v i t y. Dopamine is thought to re gu l a t e basal as well as alcohol-induced hyperlocomotion. Contrary to human studies, howe ve r, the D 2 knockout mice, which consumed less alcohol, also demonstrated reduced sensitivity to the a c t i vating effects of alcohol. On the other hand, mice lacking the D 4 re c e p t o r we re supersensitive to alcohol-induced locomotion; to date, these mice have not been tested for alcohol consumption (Rubinstein et al. 1997).
In contrast with the decrease in alcohol drinking observed in dopamine receptor knockout mice, initial studies of mice lacking the 5-HT 1 B re c e p t o r s h owed that they consumed twice as much alcohol as wild-type control mice ( Crabbe et al. 1996). Howe ve r, the i n c reased consumption demonstrated by the mutant mice was not re p l i c a t e d in later studies. This is most likely attributable to latent genetic backg round interactions between the ES cell genotype and the genotype of the i n b red strain on which the mutation was bred. This interaction did not appear until after several generations of bre e ding had been conducted (Crabbe et al. 1999). Additional tests of alcoholinduced behaviors indicated that initial sensitivity to the ataxic effects of alcohol was also increased in 5-HT 1 B mutants, whereas tolerance to chro n i c t reatment developed more slowly and m e a s u res of withdrawal sensitivity we re not affected by the deletion of the gene. These results are similar to those re p o rt e d for the ove re x p ressing 5-HT 3 t r a n s g e n i c s described earlier. The re s e a rch conclusions indicating that both undere x p re s s i n g and ove re x p ressing 5-HT re c e p t o r mutants would have similar phenotypes may appear counterintuitive; howe ve r, the 5-HT receptors are hetero g e n e o u s and have different functions in the brain. For example, 5-HT 1 B re c e p t o r s regulate release of serotonin from the n e u ron, whereas activation of 5-HT 3 receptors produces a rapid excitation of the neuron. Nu m e rous other studies h a ve re p o rted the effect of single-gene mutations on alcohol drinking, indicating that many genes regulate this phenotype. ( See the table for re f e rences to t h e s e and other receptor protein knockout s t u d i e s . ) Alcohol re s e a rch with knockout mice has also targeted certain enzyme proteins known as kinases. Protein kinases a re enzymes that activate or deactiva t e the function of proteins, including receptor proteins, by attaching phosp h a t e g roups to the proteins. For example, protein kinase C (PKC) may modi f y, and thus affect the function of, the G A B A A re c e p t o r. The activation and d e a c t i vation of specific proteins are two components of a signaling mechanism t h rough which chemical signals are re l a yed from the cell's surface to its interior. A mutation to create a defective (-) gene is introduced.
The (-) gene is introduced into embryonal stem (ES) cells in tissue culture; not all ES cells will incorporate the (-) gene into their DNA.
Cells are grown in a medium that allows only cells with the (-) gene to multiply.
Cells with the (-) gene are injected into mouse embryos.
Embryos develop into chimeric mice expressing the (-) gene in some cells and the (+) gene in other cells; one of the chimeric mice is mated with a normal mouse.
Mouse pups (i.e., F 1 animals) carrying a (+) and a (-) gene copy are identified and mated with each other.
The offspring (i.e., F 2 animals) are analyzed; about 25 percent will have inherited the (-) gene from both parents and will completely lack the (+) gene.
Re s e a rch suggests that PKC function is associated with alcohol's effects on the brain (Stubbs and Slater 1999). P KC is comprised of a family of enzyme subtypes (Pa rker 1994). Re c e n t l y, knockout models of two of these subtypes have been tested for alcohol behaviors. PKCγ is exc l u s i vely expre s s e d in the brain, including regions associated with alcohol sedation and ataxia. Tests of initial sensitivity to the sedating effects of alcohol demonstrated that mice lacki n g P KCγ we re less sensitive than wildtype control mice. This may be re l a t e d to PKCγ's action at the GABA A re c e p t o r, as alcohol-enhanced inhibition of the receptor function was absent in brain tissue from the null mutant mice comp a red with the wild-type controls (Ha r r i s et al. 1995). In addition, rapid and c h ronic tolerance to alcohol was decre a s e d in these mice, albeit dependent on b a c k g round genotype (Bowers et al. 1999(Bowers et al. , 2000a. PKCε is also highly e x p ressed in the brain and is localize d in some but not all of the same brain regions as PKCγ. Tests of alcohol c o nsumption have indicated that mice lacking PKCε i n c re a s e d their drinking 200 p e rcent over that of control mice and, in contrast with PKCγ mutants, demons t r a t e d an i n c re a s e d sensitivity to alcoholinduced sedation (Hodge et al. 1999).
Tests of alcohol sensitivity in kinase knockout mice are not limited to the P KC family of enzymes. Re c e n t l y, re s e a rch has been conducted using the f o l l owing types of mice: mice lacking the protein kinase A re g u l a t o ry subunit βII (i.e., PKAβII), mice lacking the t y rosine kinase Fyn (i.e., a kinase of the amino acid tyrosine), dopamine beta h yd roxylase knockout mice (i.e., mice lacking the dopamine beta form of the h yd roxylase enzyme), and mice lacking forms of the gene for aldehyde dehyd rogenase (i.e., an enzyme invo l ved in alcohol metabolism) (see table).
From these investigations of knockout and transgenic mice, re s e a rc h e r s can easily appreciate the complexity of a l c o h o l's actions and the difficulty in d e f i n i t i vely interpreting the contributions of individual proteins to alcoholism. The examples discussed in this a rticle are by no means an exhaustive re p resentation of all mutant mouse models used in alcohol re s e a rch (see table). The techniques of re verse genetics continually evo l ve, and as new models are developed, scientific and clinical understanding of the neurobiology of alcoholism will most certainly continue to advance. s